Novel hydroxy naphthol blue-based recombinase polymerase amplification for the rapid detection of Staphylococcus pseudintermedius

Staphylococcus pseudintermedius (S. pseudintermedius) is a significant cause of pyoderma, soft tissue, urinary tract, and ear infections in cats and dogs. Bacterial culture and biochemical phenotypic assays are the gold standards for clinical diagnosis of bacteria but molecular methods have now been developed to identify and differentiate S. pseudintermedius. However, these methods require complex and expensive equipment, restricting usage in many laboratories. This study developed a rapid, specific, and sensitive detection method for S. pseudintermedius based on recombinase polymerase amplification (RPA) combined with hydroxy naphthol blue (HNB) dye. The RPA primer was designed, optimized, and subjected to amplify the spsL gene as the molecular diagnostic target. HNB dye was added to the reaction, successfully enabling the development of a colorimetric detection method for S. pseudintermedius. This RPA-HNB assay completed amplification at 37 °C within 20 min, with results visualized by the naked eye as a color change. All processes were completed within an hour. The assay showed the limit of detection at 1 copy per reaction, with sensitivity and specificity against S. pseudintermedius 0.98 and 1.00, respectively. Results indicated that our assay provided high sensitivity and specificity for detecting S. pseudintermedius with reduced detection time.